菊糖蔗糖酶的性质鉴定及菊糖的酶法合成

菊糖作为益生元和膳食纤维,具有许多重要的生理功能,广泛应用于食品、医药等领域.微生物菊糖蔗糖酶可以以蔗糖为底物合成较植物菊糖具有更高分子量的菊糖.文中通过基因数据库筛选获得一段拟表达菊糖蔗糖酶的基因.通过N-端和C-端截断的方式,保留中间催化域,构建重组质粒.将重组质粒在大肠杆菌表达系统中表达,粗酶液经Ni2+亲和层析...     

细菌的核酮糖单磷酸途径与甲醛同化作用

核酮糖单磷酸途径最初在甲基营养菌中发现,现在被认为是在细菌中广泛存在的和甲醛同化作用及脱毒相关的一条途径,该途径的关键酶是6-磷酸己酮糖合成酶和6-磷酸己酮糖异构酶。文章将介绍来源于各种细菌的核酮糖单磷酸途径的生理作用及其两个关键酶基因的组织结构、表达调控机制与应用前景。     

木糖异构化木酮糖的制取及分离纯化

木糖是仅次于葡萄糖的重要生物工程生产基质,但不能直接被包括啤酒酵母在内的大多数酵母利用,而其异构体——本酮糖可以被这些微生物代谢。本研究利用木糖异构酶使木糖异构化生成木酮糖,在50℃和初始pH=8.0的条件下,木酮糖平衡转化率为21.6％,且随反应温度升高而增大。应用液相色谱技术使木酮糖与木糖分离,经三级色谱分离,纯木酮糖的精制回收率达43.8％。     

木酮糖激酶表达水平对酿酒酵母木糖代谢产物流向的影响

在酿酒酵母中分别引入真菌和细菌的木糖代谢关键酶,木糖还原酶基因XYL1、木糖醇脱氢酶基因XYL2和木糖异构酶基因xylA. 并在此基础上以共转化策略超表达下游关键酶木酮糖激酶基因XKS1. 与亲本菌株相比,用pMA91和YEp24质粒表达XKS1的重组菌株,木酮糖激酶（xylulokinase,XK）活性分别提高了14和6.7倍. 在限氧条件下,重组菌株对木糖和葡萄糖的共发酵结果显示,表达XYL1,XYL2以及XKS1的重组菌株HSXY-251木糖消耗为12.4 g/L,提高了120.9%,乙醇产量达到9.4 g/L,提高了36%,副产物木糖醇产量为0.7 g/L,下降了84.9%.     

大肠杆菌转酮醇酶分子改造及催化酒石酸半醛合成

转酮醇酶(transketolase, TK, EC. 2.2.1.1)是一种焦磷酸硫胺素和二价阳离子依赖性酶,可催化二碳单位的转移,可逆形成C–C键,在多酶催化生产化学品、药物前体和不对称合成方面有广泛应用。文中以大肠杆菌(Escherichia coli) K12转酮醇酶TKTA为研究对象,通过定点饱和突变和组合突变提升对非磷酸化底物的反应活性,并探索突变酶TKTA＿M催化合成酒石酸半醛。结果表明：突变酶TKTA＿M (R358I/H461S/R520Q)最适反应温度为32℃,最适反应pH为7.0,以D-甘油醛为受体底物的比酶活为(6.57±0.14) U/mg,是野生型比酶活((0.71±0.02) U/mg)的9.25倍。在酶学性质研究的基础上,设计20 mL的反应体系,以50 mmol/L 5-酮基-D-葡萄糖酸和50 mmol/L非磷酸化乙醇醛为底物,TKTA＿M催化合成酒石酸半醛,最终酒石酸半醛的产量为3.71g,摩尔转化率为55.34%。研究结果为生物质制备L-(+)-酒石酸提供数据支撑,同时为转酮醇酶催化非磷酸化底物提供了借鉴。     

蔗糖异构酶催化生产异麦芽酮糖研究进展

作为蔗糖的一种异构体,异麦芽酮糖具有许多独特的生理功能,例如非致龋齿性、益生元特性、适合糖尿病人使用以及对大多数细菌和酵母的抗性等,因而受到广泛关注。异麦芽酮糖主要是通过蔗糖异构酶催化蔗糖转化形成,反应中同时生成的海藻酮糖以及少量的葡萄糖和果糖,给工业生产带来困扰。简要论述异麦芽酮糖的特性、生理功能及其生产中存在的问题,重点论述蔗糖异构酶催化蔗糖转化的机理,为异麦芽酮糖在食品工业中的开发利用提供参考。     

酶法生产稀少糖的研究进展

稀少糖是指自然界中存在稀少的单糖及其衍生物,可应用于食品、制药和营养等许多领域,同时也可以作为各种天然产品和候选药物的原料。目前大多数稀少糖因其在自然界中含量稀少而非常昂贵,而且这些稀少糖的化学合成原料价格高昂,不利于其大量生产。由于酶催化反应具有反应条件温和、特异性强、效率高、可持续性强等优点,稀少糖酶法生物合成已成为这一领域的有力工具。文中就稀少糖,包括D-阿洛酮糖、D-塔格糖、D-山梨糖、L-果糖、D-阿洛糖等其他稀少糖的生物学功能及应用,以及稀少糖相关酶的研究和酶法生产进行综述。     

木酮糖激酶基因整合表达载体构建及在酿酒酵母中的过表达

【目的】以载体p406ADH1为构建骨架,构建一个酿酒酵母(Saccharomyces cerevisiae)工业菌株的整合表达载体。【方法】通过酶切连接的方式,将4个元件片段:作为筛选标记的G418抗性基因KanR,用于基因表达的ADH1终止子片段,酿酒酵母W5自身木酮糖激酶基因,18S rDNA介导的同源整合区,插入到骨架质粒p406ADH1中,得到多拷贝整合表达载体pCXS-RKTr。将该载体线性转化酿酒酵母后,对转化子中木酮糖激酶酶活进行测定,检测其表达情况。【结果】重组质粒在酿酒酵母体内实现了木酮糖激酶的高水平稳定表达,其酶活力是初始菌株的2.87倍。【结论】本实验构建了一个酿酒酵母工业菌株整合表达载体,并用此载体过表达了其自身的木酮糖激酶基因。该重组质粒载体的构建可以有效解决酿酒酵母中自身木酮糖激酶酶活较低的情况,这为利用木糖高产乙醇酿酒酵母基因工程菌株的构建和其它酵母重组质粒载体的构建奠定基础。     

非水相体系酶催化反应研究进展

非水相酶催化反应是酶催化反应中的一个重要方面。非水相溶剂通常可增加底物溶解度,减少水相中的副反应,加快生物催化的速率和效率,在药物及药物中间体和食品等方面具有较大的应用价值。以下探讨了非水相体系对酶活力及酶促反应速率的影响因素,并阐述酶的化学修饰、固定化及定点突变对酶活力的影响,进一步分析无溶剂系统、反胶束、超临界流体及离子液体的不同溶剂体系对酶反应速率及催化效率的影响。此外,还列举一些非水相酶催化反应的应用实例。     

Structural and kinetic studies of induced fit in xylulose kinase from Escherichia coli

The primary metabolic route for D-xylose, the second most abundant sugar in nature, is via the pentose phosphate pathway after a two-step or three-step conversion to xylulose-5-phosphate. Xylulose kinase (XK; EC 2.7.1.17) phosphorylates D-xylulose, the last step in this conversion. The apo and D-xylulose-bound crystal structures of Escherichia coli XK have been determined and show a dimer composed of two domains separated by an open cleft. XK dimerization was observed directly by a cryo-EM reconstruction at 36 A resolution. Kinetic studies reveal that XK has a weak substrate-independent MgATP-hydrolyzing activity, and phosphorylates several sugars and polyols with low catalytic efficiency. Binding of pentulose and MgATP to form the reactive ternary complex is strongly synergistic. Although the steady-state kinetic mechanism of XK is formally random, a path is preferred in which D-xylulose binds before MgATP. Modelling of MgATP binding to XK and the accompanying conformational change suggests that sugar binding is accompanied by a dramatic hinge-bending movement that enhances interactions with MgATP, explaining the observed synergism. A catalytic mechanism is proposed and supported by relevant site-directed mutants.     

Enzymatic regioselective synthesis of sucrose acrylate esters

Using a protease (at 100 g l^–1) from Bacillus licheniformis, enzymatic acryloylation of sucrose (1 M) with vinyl acrylate (4 M) was carried out in anhydrous pyridine and yielded sucrose acrylate esters with more than 90% of sucrose converted in 24 h. After 5 days of reaction, the ester products consisted of 70% sucrose monoacrylate and 30% sucrose diacrylate. The monoester product was a sucrose 1-acrylate and the diester products consisted of sucrose 6,1-diacrylate and sucrose 6,1-diacrylate in the ratio of 3:2.     

Effects of borate on isomerization and yeast fermentation of high xylulose solution and acid hydrolysate of hemicellulose

d-Xylose has been isomerized by immobilized d-glucose isomerase (EC nomenclature is now d-xylose isomerase, d-xylose ketol-isomerase, EC 5.3.1.5; EC 5.3.1.18 is a deleted EC entry). Temperature has a profound influence on the equilibrium concentration of d-xylulose. When 1 md-xylose was isomerized in the presence of various concentrations of borate, maximum conversion (80%) was observed at 0.2 m sodium tetraborate. Temperature (40–69°C) and pH (6.0–7.5) had an insignificant effect on the equilibrium when borate was present. d-Xylose (0.5 m) was isomerized by d-glucose isomerase in the presence of various concentrations of sodium tetraborate (0.0125–0.25 m). Based on the initial rate of ethanol production and the fraction of total sugar converted into ethanol after 24 h of yeast fermentation, an optimum tetraborate concentration of 0.05 m was determined for both isomerization and fermentation. At an acidic pH, the rate of fermentation was faster than at neutral pH when borate was included in the d-xylose—d-xylulose system. Acid hydrolysate of bagasse hemicellulose could not be fermented at a pH lower than 5. Therefore, a compromise condition, pH 6.0, was chosen for fermentation.     

Enzymatic synthesis of polyphenols from highly phenolic lignin-based polymers (lignophenols)

Peroxidase-catalyzed polymerization of lignin-based macromonomers (lignophenols), lignocatechol and lignocresol, prepared by phenolation of lignin with catechol or p-cresol, was carried out in aqueous organic solvent mixtures. The two lignophenols were polymerized to give cross-linked polymers. The highest yield of polymerization (83%, w/w) was obtained with lignocatechol, and the maximum yield for the polymerization of lignocresol was 55% (w/w). Pyrolysis GC-MS analysis of polymers indicated that the polymerization of lignophenols involved the oxidative coupling of the introduced phenol derivatives.     

防腐剂DMDMH合成中游离甲醛含量的控制

在DMDMH的合成中加入尿囊素,采用一次性合成方法,可减低产品中游离甲醛额含量,控制游离甲醛含量≤0.1%,产品收率达到90%以上,稳定性达到生产要求。     

生物催化甲醛生成L-木糖

在当今不可再生资源日益消耗的情形下,利用生物合成的技术,将甲醛转变成糖类,具有重要意义。该过程最重要的是找到一个合适的催化剂组合来实现甲醛的二聚反应。在最近的研究中,报道发现了一种乙醇醛合酶(Glycolaldehyde synthase,GALS)可以催化这一反应,将其与D-果糖-6磷酸醛缩酶(D-fructose-6-phosphate aldolase,FSA)组合使用,即"一锅酶"法,可以利用甲醛合成L-木糖,并且转化率可达64%。这一过程的实现也为合成其他糖的反应提供了参考。     

应用乙酰短杆菌酶法合成2'-脱氧腺苷

胸苷和腺嘌呤在乙酰短杆菌作用下转化成胸腺嘧啶和目标产物2’-脱氧腺苷。50mL的三角瓶装液10mL,40mmol/L的底物,50mmol/L的磷酸盐缓冲液(pH7)进行反应,5%游离乙酰短杆菌在55℃下,经1h转化率就可达到65.6%。